> Hi Bryan,
> I know that most people say that destaining is necessary for EtBr
> detection of DNA in agarose gels. We NEVER destain! It is absolutely not
> necessary, and we consider it wasted time. The problem might be that most
> people use WAY to much EtBr, so they have to get rid of the excess
> afterwards. Thats how we do it:
>> *run gel IN ABSENCE of EtBr; we use TAE buffer
> *stain for 15min at RT, with EtBr diluted 1:10.000 from a 1% stock
> solution. We dilute in water, but if it could be necessary to return your
> gel to the tank and let it run longer, dilute in electrophoresis buffer.
> You will not see the orange color of EtBr in this dilution, but still its
> more than enough to give you a sensitivity down to 5-10ng of DNA (on a
> 6x8cm minigel). Also, the staining solution can be re-used at least
> 10times, without affecting the sensitivity. For storage, keep it in a
> brown glass.
> *after 15min, rinse briefly with water (10sec is enough) end directly look
> at the gel on your illuminator: You´ll see orange bands on a black
> background...
>> SURE: that sensitivity is neither "the ultimate sensitivity " nor "lower
> than 0.1ng DNA", but in routine work you almost never have such low
> amounts (Why should you, in the age of cloning and PCR?).
>> OK: there´s one application where destaining IS necessary: the
> electrophoresis of RNA on formaldehyde-containing gels.
Hello Frank:
Thanks for the outline of your "no destaining method", I will certainly
experiment with this approach, when applicable. There is a historical
reason (of course) for our destaining of acrylamide gels. This is due to
the fact that many of our diagnostic PCR products have been quite short
(55 to 110 bp). Our products were from allele-specific PCRs (go or no go
dependent on match or mismatch at the 3' ultimate and/or penultimate
base in the primer) but the alleles were simply site-specific mutants
(eg. ras) that often are relatively weak products since the mutant, if
present, is often a minor fraction of the normal allele. We had learned
the hard way that excess time spent either in staining or in destaining
could quite rapidly result in loss of signal due to elution of these
small DNA pieces out of the gel. So the ad hoc solution was to use
relatively strong EtBr (stain 1 minute) and follow up with a 3 to 5
minute destain in water or buffer. The background stain remaining from
this procedure is low enough to permit good polaroid 3000 photos with 3
to 4 seconds or more exposure. Recently I have "discovered" (no doubt
reinventing someone's tried and true wisdom) that this "elution loss"
was an unnecessary concern, an artifact associated with habitual use of
5 or 6% polyacrylamide gels-- For example in current work I now find
that I can have good retention of very small pieces of DNA within gels
by using higher percentage acrylamide, allowing for nice retention in
stain or destain for 10 minutes or more, probably permitting longer
staining with low concentration EtBr, much as you suggest (I'll try it).
My guess is that the allele-specific PCRs with 55 bp products could have
been nicely stained with a low concentration EtBr if run on say a 10-12%
polyacrylamide.
Thanks for the informative discussion.
-Bryan